The classic path to protein biomarker discovery is by measuring differential levels of proteins in blood or tissues of interest. This approach has not been a great success, mainly because the complexity of the blood proteome precludes detection of proteins or peptides at low ng/mL levels without time-consuming pre- fractionation. This status quo may end by either improving existing markers or by employing discovery and validation schemes that are conceptually novel. To this end, we will develop an innovative screening and verification platform using novel yet robust approaches to measure protease activities in blood. The idea behind this approach originated in an observation that protease activities uniquely shape the serum peptidome to provide class discrimination between patients with different types of solid tumors. This model is further supported by a wealth of peer-reviewed literature describing gene expression, protein levels and enzymatic activities of aminopeptidases, and other proteases, in solid tumors and blood. It presents opportunities for biomarker discovery and validation approaches that are unique by today's standards. We have previously developed a series of fluorescence-based activity assays to analyze individual proteases, as opposed to the collective activities measured in the past, that appear unbalanced in blood of cancer patients. Fluorescent assays have advantages over most conventional protein measurements, including low cost, parallel high- throughput analysis, ease of use, portability, and being fully transparent to the entire blood proteome. By putting an antibody (Ab)-mediated affinity-capture step on the front, a technique termed Immobilized Protease Activity Test (IPAT), analytical specificity and sensitivity are significantly improved. We propose to procure and test critical reagents to develop, optimize and implement sensitive IPAT assays for 18 aminopeptidases and 14 selected other proteases (e.g., MMPs, cathepsins, caspases, etc). This will allow measuring quantitative changes of their activities in blood as a result of cancer. IPAT technology will be implemented in two configurations: (1) Abs will be immobilized to magnetic nanoparticles and all steps automated in a robot ('bead- IPAT'). (2) Abs immobilized in 96-well micro-titer plates ('well-IPAT') in a configuration similar to an ELISA. The resulting set will be tested in two clinically relevant biomarker discovery studies. First, to evaluate the utiliy of plasma protease assays as a means of improving specificity of radiographic screening for breast cancer, we will assess its performance in predicting the outcome of breast biopsies. Second, we will examine the value of serum IPAT assays as a prognostic factor for survival in patients with progressive metastatic castration- resistant prostate cancer receiving chemotherapy. The clinical objective is to combine the data with existing, though imperfect markers and develop a prognostic tool that accurately gives the probability of patient survival. This application is innovative as it is the first time a class of enzymes, undetected in past proteomic screens, will be targeted as potential activity biomarkers. If our developments are successful and, as a result, effective functional cancer biomarker panels emerge, it would have a major clinical impact.